It is known that the cause of a great part of the dermatological diseases (psoriasis, malignous and benign tumors) is the irregularly enhanced proliferation of epidermal cells. Though several cytostatic drugs are available, their common feature and drawback is that they are toxic and are not specific for the cells of the organ or tissue where the irregular proliferation occurs.
The existence of endogenous substances specifically inhibiting the cell proliferation was supposed at the beginning of the 1960s when tests were carried out in order to determine how the organs of the adult humans and animals can keep their constant size. The compounds ensuring the equilibrium between the inhibition and proliferation of cells were called chalones A-4762-67/539 KY (Biol. Rev., 37, 307 (X1962X).
The chalones are produced in the cells of tissues, thus they paracrinally exhibit their effect by the so-called negative feed-back mechanism. Since then these substances have been isolated from various tissues (mouse skin, rat medulla, human leucocytes, mouse liver, mouse intestine) and the tests carried out on them seem to support the above chalon-hypothesis: the cell proliferation inhibiting effect of these compounds is strictly specific and reversible.
Today the chemical structure of the numerous inhibitors of chalone type is known. All of these substances are peptides.
The structure of the epidermal inhibitor pentapeptide (EI) was discovered in the middle of the 1980's (Cell. Biol. Int. Rep. 8, 379 (1984); Biological Regulation of Cell Proliferation, Eds. R. Baserga et al., Raven Press, N.Y. 1986, Vol. 34, page 259). The amino acid sequence of this peptide is as follows: EQU Xaa Glu Asp Ser Gly Sequence No. 2
wherein Xaa is pyroglutamic acid.
Both the natural and synthetic peptides inhibit the epidermal cell proliferation of mice even in a very small dose (10.sup.-14 to 10.sup.-11 mol/animal). In vitro it reduces the proliferation rate of mouse epidermal cells by about 40% when administered in a dose of 10.sup.-12 to 10.sup.-8 mol/l.
It is evident that this compound potentially may be a drug for the specific treatment of dermatological diseases caused by epidermal cell proliferation and tumors of other epidermal origin.
The data of structure-activity relationship available up to now show that the activity is highly connected with the structure of EI.
However it was found that the replacement of the N-terminal pyroglutamic acid of the peptide by pyro-alpha-aminoadipic acid had not influenced the cell proliferation inhibiting activity thereof.
The change of pyroglutamic acid to pyro-alpha-amino adipic acid in biologically active peptides is not unprecedented in the prior art. Generally this substitution results in keeping of the biological activity or--in certain cases--some increase of the activity (German published patent application No. 2,514,381, Hungarian patent No. 180,926 and German patent No. 3,226,242; Life Sci., 34, 2597 (1984).
However, an even more significant advantage of the peptide analogues obtained in this manner is that they are less sensitive to peptide degrading enzymes, thus their effect is more durable.
Namely, it is known that the main route of metabolism and inactivation of pyroglutamyl peptides is the splitting off of the pyroglutamyl moiety by the pyroglutamyl-aminopeptidase enzyme. The peptides containing a N-terminal pyro-alpha-aminoadipic acid are resistant to this enzyme.
From these facts, the difference in the strength and duration of the activity, i.e. the inhibition of the proliferation of the epidermal but malignous (so-called HeLa) cells, by the pyro-alpha-aminoadipyl analogoue of the invention, as compared to the known peptide, is surprising and preferable from practical points of view.
The inhibitory effect of the compound of the present invention on the proliferation of tumorous cells is twofold compared to the natural peptide and this effect is maintained even after 13 hours. Considering that the proliferation of healthy epidermal cells is inhibited by both peptides in the same degree, the inhibiting effect of the peptide of the invention is significantly more selective than that of the known one. The increase of selectivity could not have been expected in the knowledge of the prior art.
The novel peptide of sequence No. 1 can be prepared by any method conventionally used in peptide chemistry. For the synthesis of the peptide the stepwise strategy and the benzyl/tert-butyl-type protecting group combination were preferably employed.
This latter means that the alpha-amino groups are temporarily protected by protective groups of benzyl type which can selectively be removed by hydrogenolysis, while the permanent protection of other functional groups is ensured by protective groups of t-butyl type which are split off at the end of the synthesis in one step by acidolysis.
According to a preferred embodiment of the process glycine-t-butyl ester is condensed with N-benzyloxycarbonyl-O-t-butyl-L-serine to form a protected dipeptide of formula Z-Ser(.sup.t Bu)-Gly-(O.sup.t Bu) in the presence of dicyclohexyl carbodiimide, from which the amino protective group is removed by catalytic hydrogenation.
The free dipeptide ester obtained is acylated with N-benzyloxycarbonyl-L-asparaginyl-alpha-(1-hydroxy-succinimidyl)-beta-(t-b utyl)-ester to receive the protected tripeptide of formula Z-Asp(O.sup.t Bu)-Ser(.sup.t Bu)-Gly-O.sup.t Bu.
The benzyloxycarbonyl protective group is removed by hydrogenolysis again, the amino group liberated is acylated by -benzyloxycarbonyl-L-glutamic acid-alpha-(1-hydroxy-succinimidyl)-gamma-(t-butyl)-ester.
The protected tetrapeptide of Sequence No. 3 which is Glu Asp Ser Gly wherein the gamma glutamyl and beta aspartyl carboxy groups are esterified by t-butyl, the serine hydroxy group is etherified by t-butyl, the glycine carboxy group is esterified by t-butyl, and the glutamic acid amino terminal is blocked by benzyloxy carbonyl is subjected to catalytic hydrogenation and the free tetrapeptide ester thus obtained is acylated with L-pyro-alpha-aminoadipic acid-pentafluorophenyl ester.
From the protected pentapeptide the t-butyl groups are removed in one step by trifluoroacetic acid treatment, thus the novel compound of sequence No. 1 is obtained which is purified by recrystallization from aqueous alcoholic medium. The compound thus obtained is suitable for pharmaceutical use.
In the in vitro test of the cell proliferation inhibiting effect of the new compound of the invention, normal diploid Chang and epidermal HeLa-S3 tumor cell lines were used.
In order to verify the specificity of the effect, the tests were also carried out by using non-target rat medulla and rat thymus cell lines.
The effect on cell proliferation was tested by measuring the incorporation of labelled thymidine.
The in vitro toxicity of the new compound was examined by .sup.51 Cr emission test.
The cell cultures were developed as follows: